Cancer develops from cells that acquire the ability to proliferate despite several biochemical control mechanisms that suppress growth. Proliferation of cells leads to the formation of a tumor composed of heterogeneous groups of cells exposed to different microenvironments. As the tumor grows, blood vessels infiltrate the mass randomly which causes areas of the tumor to be deprived of nutrients and oxygen. It has been shown that tumors with a preponderance of hypoxic (low oxygen) regions are less sensitive to standard chemo- and radiotherapy. This may be due to the fact that under hypoxic conditions, cells stop dividing, and are thus less sensitive to traditional therapies that target actively dividing cells. This project is designed to investigate the biochemical mechanism of the cellular response to hypoxia in order to create the foundation for the development of treatment strategies that target these cells. The mechanism of hypoxia-mediated cell proliferation arrest will be examined by analysis of key cell division cycle regulatory proteins in nontransformed CV-1P and ovarian cancer cells maintained under hypoxic conditions. The role of the tumor suppressor protein, Retinoblastoma (pRb), as well as other pocket proteins, (p107 and p130) in hypoxia-mediated cell proliferation arrest will be investigated. Under hypoxic conditions, pRb becomes activated (dephosphorylated) by the concerted action of a decrease in pRb-specific kinase activity coupled with an increase in pRb-specific phosphatase activity. The mechanisms by which these enzymes are regulated in hypoxia will be examined. In addition, pRb exerts its activity as a tumor suppressor by binding to and inhibiting specific cellular proteins. Therefore, the protein binding activity of pRb in cells maintained under hypoxic conditions will be investigated. Finally, the function ofp 107 and p130 in hypoxia-mediated cell proliferation arrest will be examined. The performance of the studies described in this proposal will serve as a basis to build upon in future studies directed toward increasing the efficacy of cancer treatment.